Abstract: 【Objective】The objective of this study is to clone barnyardgrass (Echinochloa crus-galli) 1-aminocyclopropane-1- carboxylate oxidase gene (EcACO), analyze its expression and test its enzyme activity, and to unravel the quinclorac-resistant mechanism of E. crus-galli to quinclorac.【Method】The partial sequence of EcACO obtained from E. crus-galli transcriptome pyrosequencing was used to design primers for cloning EcACO from quinclorac-resistant and susceptible E. crus-galli. EcACO was then cloned and sequenced. The nucleotide and putative amino acid sequence analysis were compared using DNAman and GenDoc softwares. The transcript levels of EcACO between resistant and susceptible biotype E. crus-galli were determined by real-time quantitative PCR (qRT-PCR) with β-actin gene as the reference. Finally, the open reading frame (ORF) sequences of EcACO from resistant and susceptible biotypes E. crus-galli were inserted into the expression vector pMAL-c5x, respectively. After the recombinant plasmids were transformed into Escherichia coli strain BL21, the fusion proteins were expressed by the induction with 0.4 mmol·L^-1 IPTG for 16 h at 18℃. The soluble proteins were purified with MBP column for the measurement of ethylene released from MBP::EcACO fusion protein. 【Result】EcACO was isolated from E. crus-galli with quinclorac-resistant and susceptible biotypes of E. crus-galli. The ORF of EcACO was 936 bp, encoding 311 amino acids, with pI 5.4 and Mw 35 kD. The deduced amino acid sequences shared high identity with other ACO sequences from Setaria italica (93%), Zea mays (92%) and Sorghum bicolor (91%). Compared with EcACO from the susceptible biotype, five site mutations of EcACO were found in the resistant biotype, of which three site mutations were located in the putative conserved domain. Furthermore, qRT-PCR results showed that there was no significant difference in expression level of EcACO between resistant and susceptible biotype. Using the prokaryotic expression system and the measurement of MBP::EcACO activity, the released amount of ethylene in the MBP::EcACO from susceptible biotype was 2.15 folds higher than that from resistant biotype.【Conclusion】EcACO was identified from quinclorac-resistant and susceptible E. crus-galli. Compared with the susceptible biotype, the EcACO from the resistant one had five amino acid mutations, of which three site mutations were in the conserved domain. This might probably contribute to the reduction of released amount of ethylene and result in quinclorac resistance of E. crus-galli.

Key words: Echinochloa crus-galli, 1-aminocyclopropane-1-carboxylate oxidase, gene cloning, site mutation, expression